Gliomas, the most frequent primary brain tumors, exhibit very poor prognosis which is largely due to their highly infiltrative nature. Genetic and biochemical studies have demonstrated that gliomagenesis involves stepwise accumulation of genetic alterations of signal transduction pathways and cell cycle related proteins. Indeed, signaling pathways such as Ras/Erk/AKT and pathways coupled to diacylglycerol (DAG) production are highly active in glioma cells. RasGRP3 is a guanine nucleotide exchange factor (GEF) that activates small GTPases including Ras and Rap1. RasGRP3 is activated by DAG which is generated by phospholipase Cg downstream of activated growth factors receptors in glioma cells. The activation of RasGRP3 is controlled by membrane recruitment through its DAG binding C1 domain. Our preliminary results demonstrate that RasGRP3 is expressed in gliomas and regulates glioma cell migration and invasion and the activation of Ras, Rap1 and the AKT and Erk1/2 pathways. In addition, using a pull-down assay, we recently identified Arp3 as a novel interacting protein of RasGRP3 in glioma cells. Since Arp3 regulates cell migration, we hypothesized that RasGRP3 plays a major role in glioma cell migration via its interaction with this protein. The main objective of this study is therefore to delineate the mechanisms involved in RasGRP3 effects on glioma cell migration in vitro and in vivo focusing on its interaction with Arp3. Specifically, we will further characterize the interaction of the endogenous and overexpressed RasGRP3 and Arp3 using co-immunoprecipitation, immunofluorescence staining for co-localization and FRET assays for the determination of a direct interaction. Since Arp3 plays a major role in actin polymerization which leads to cell spreading and cell migration, we will determine the role of RasGRP3 and its interaction with Arp3 in these processes. We will also examine the possibility that Arp3 acts as an anchoring/scaffold protein for RasGRP3 during its translocation. Finally, we will examine the role of RasGRP3 and Arp3 in glioma cell migration in vivo using xenografts derived from U87 cells overexpressing RasGRP3 and CD133+ glioma stem cells in which RasGRP3 is silenced. The results of these studies will delineate the role of RasGRP3 in glioma cell migration focusing on its interaction with Arp3 and may provide the basis for the development of novel therapeutic approaches for the treatment of malignant glioma using RasGRP3 as a molecular target.